Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx.

Johanna Detzner,Alexander Mellmann,Helge Karch,Johannes Müthing,Hans-Ulrich Humpf,Gottfried Pohlentz,Charlotte Püttmann

doi:10.3390/ijms221810002

Johanna Detzner, Alexander Mellmann + Show 5 more

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https://doi.org/10.3390/ijms221810002

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Abstract

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.

Highlights

Among numerous gastrointestinal pathogens that cause severe gastroenteritis in humans enterohemorrhagic Escherichia coli (EHEC), the major subgroup of virulent Shiga toxin (Stx)-producing E. coli (STEC) can colonize the human gut mucosa [1,2], where EHEC are able to interact via attaching and effacing (A/E) lesions with the host colon epithelium.Such A/E lesions are characterized by intimate pathogen attachment to the apical surface of enterocytes and reorganization of the actin cytoskeleton beneath the adhered bacteria into pedestals leading to brush border and microvilli deterioration [3,4]
The locus of enterocyte effacement (LEE), a genome-inserted pathogenicity island, comprises the genes responsible for causing A/E lesions including a type III secretion system (T3SS) that encodes the adhesive protein intimin, its receptor named translocated intimin receptor (Tir), and other effector proteins being translocated by the T3SS from the bacterial cytosol into the infected cells [5,6,7,8]
We isolated Stx-binding GSLs from pHCoEpiCs cultivated without serum and performed full structural characterization of their structures

Summary

Introduction

Among numerous gastrointestinal pathogens that cause severe gastroenteritis in humans enterohemorrhagic Escherichia coli (EHEC), the major subgroup of virulent Shiga toxin (Stx)-producing E. coli (STEC) can colonize the human gut mucosa [1,2], where EHEC are able to interact via attaching and effacing (A/E) lesions with the host colon epithelium. Such A/E lesions are characterized by intimate pathogen attachment to the apical surface of enterocytes and reorganization of the actin cytoskeleton beneath the adhered bacteria into pedestals leading to brush border and microvilli deterioration [3,4]. Evidence for the involvement of Stx-containing blood cell-derived extracellular vesicles as triggering factors in HUS has been provided that dock preferentially to endothelial cells of target organs [14,15]

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